The hormonal control of glucose synthesis in the liver and kidney cortex is critical for the regulation of glucose homeostasis in all mammals. The pace setting enzyme in the pathway of gluconeogenesis is P-enolpyruvate carboxykinase (GTP) (PEPCK). The level of expression of the gene for this enzyme is sensitive to a variety of dietary and hormonal signals. We have mapped the response elements in the promoter which bind transcription factors and determined the signals which alter the rate of transcription in various nutritional and hormonal states. The PEPCK promoter (-460 to +73) contains three highly interactive regions which coordinately control transcription of the gene. Region 1 is responsible for controlling basal transcription of the gene and its response to cAMP; Region 2 contains sequences required for the liver and kidney specific expression and for thyroid hormone control of gene transcription; Region 3 has regulatory elements involved in the response of the gene to glucocorticoids and insulin. A model to explain the complex pattern of regulation of PEPCK gene transcription has been developed based on previous research. This model will be tested in detail during this granting period. We propose to determine the physiological regulation exerted on each of the regulatory elements within the three regions of the PEPCK promoter by analysis of function in transgenic mice. We will determine the mechanism responsible for the inhibition of PEPCK gene transcription caused by dietary carbohydrate and delineate between an effect caused by insulin and/or carbohydrate. In addition, the interaction of specific transcription factors involved in the control of PEPCK gene transcription will be studied, using the purified transcription factors in an in vitro binding assay, by ultra violet photofootprinting of the PEPCK promoter in vivo and by analysis of protein-protein interactions employing the yeast two-hybrid system. Mice with "knock outs" in the genes for C/EBPa and HNF-l will be analyzed to better understand the role of these proteins in controlling the tissue specific expression of the PEPCK gene in the liver and kidney. Preliminary studies suggest that C/EBPa may be critical for the development of the PEPCK gene in the liver and for the maintenance of glucose homeostasis during the perinatal period. The interactions of proteins binding to Regions 1 and 2 will be determined by "element shift" experiments and by the use of the two hybrid system. The goal of this research is to develop a system to dissect the Regions of the PEPCK promoter for detailed study by in vitro analysis. The results of this research should provide insight into the control of a key metabolic enzyme and allow the formulation of a set of metabolic principles which can be applied to other enzymes at key points in metabolic pathways. This research has clear implications for the understanding of the etiology of diabetes and the more basic processes which underlie the control of glucose homeostasis in mammals.